The long-term objectives of this application are to understand how molecular and epigenetic mechanisms control osteogenic differentiation of mesenchymal stem/stromal cells (MSCs), osteoblast function, and bone formation. MSCs are multipotent progenitor cells with self-renewal capabilities and multilineage differentiation potentials including osteogenesis, chondrogenesis and adipogenesis. Although significant progress has been made in understanding transcriptional control of MSC differentiation, little is known about how bone formation is epigenetically regulated. Histone methylation is an important process linked to the activation and repression of gene expression, thus it plays a critical role in epigenetic regulation of cell differentiation. While growing evidence indicates tht histone demethylases epigenetically regulate embryonic stem cell properties and functions, it is largely unknown what affect demethylases have on MSC differentiation and bone formation. To explore the role of demethylases in MSC differentiation, we systemically profiled the expression of histone demethylases in BMP-stimulated MSCs from bone marrow, as BMPs are potent inducers of osteogenic differentiation. We found that BMPs rapidly induced the expression of the lysine (K)-specific demethylase (KDM4B; also known as JMJD2B) that demethylates trimethylated histone H3 at lysine 9 (H3K9me3). H3K9me3 is a hallmark for gene silencing involved in growth and development. In general, a group of specific genes are activated when the stem cell differentiation program is triggered. Our preliminary studies demonstrated that KDM4B promoted osteogenic differentiation of MSCs while inhibiting adipogenic differentiation. Moreover, we found that the expression of Kdm4b was significantly down regulated in MSCs isolated from aging mice compared to young mice. Co-incidentally, H3K9me3 marks were significantly increased in osteoblasts of aging mice or ovariectomized mice. Based on these novel discoveries, in this application, we hypothesize that erasing H3K9me3 marks by KDM4B plays integral roles in osteogenic differentiation of MSCs in vitro and bone formation and in vivo. Three specific aims are proposed to test our hypothesis. Aim 1 is to determine whether KDM4B epigenetically regulate MSC lineage commitment through induction of DLX5. Aim 2 is to explore how erasing H3K9me3 by KDM4B coordinately regulates osteogenic differentiation of MSCs. Aim 3 is to determine whether KDM4B is required for bone formation in vivo and whether dysregulation of KDM4B impairs osteoblast function and bone formation in osteoporosis. Since histone demethylases are chemically modifiable, KDM4B may present as a novel therapeutic target for specifically controlling the differentiation of MSCs in regenerative medicine, and also lead to clues for new treatment in metabolic bone diseases such as, osteoporosis.